The present invention relates generally to compositions and methods with research and pharmaceutical applications, and more specifically, to methods for producing a retroviral vector composition of very high titer.
Since the discovery of nucleic acids in the 1940s and continuing through the most recent era of biotechnology, substantial research has been undertaken in order to affect the course of a disease through interaction with the nucleic acids of living organisms. Most recently, a wide variety of methods have been described for altering or affecting genes within humans or animals, by directly administering to the human or animal a nucleic acid molecule which alters or effects the course of a disease. In this regard, many different vectors have been utilized to deliver nucleic acid molecules to a human or animal, including for example, viral vectors derived from retroviruses, adenoviruses, vaccinia viruses, herpes viruses, and adeno-associated viruses (see Jolly, Cancer Gene Therapy 1(1):51-64, 1994).
One gene therapy approach which has shown particular promise are recombinantly produced, retroviral vector particles. Briefly, retroviruses are RNA viruses which can replicate and integrate into a host cell""s genome through a DNA intermediate. This DNA intermediate, or provirus, may be stably integrated into the host""s cellular DNA.
Although retroviruses can cause disease, they also have a number of properties that lead them to be considered as one of the most promising techniques for genetic therapy of disease. These properties include: (1) efficient entry of genetic material (the vector genome) into cells; (2) an active efficient process of entry into the target cell nucleus; (3) relatively high levels of gene expression; (4) minimal pathological effects on target cells; and (5) the potential to target particular cellular subtypes through control of the vector-target cell binding and tissue-specific control of gene expression. In using a retrovirus for genetic therapy, a foreign gene of interest may be incorporated into the retrovirus in place of normal retroviral RNA. When the retrovirus injects its RNA into a cell, the foreign gene is also introduced into the cell, and may then be integrated into the host""s cellular DNA as if it were the retrovirus itself. Expression of this foreign gene within the host results in expression of foreign protein by the host cell.
One issue however, that has arisen in developing commercial grade quantities of therapeutic retroviruses, is the ability to make sufficient retroviral vector particles at a suitable concentration to: (1) treat a large number of cells (e.g., 108-1010); and (2) manufacture vector particles at a commercially viable cost.
The present invention provides methods for obtaining high titers of retroviral vector particles at a commercially feasible cost, and further provides other related advantages.
Briefly stated, the present invention provides compositions and methods for producing high titer retroviral vector particles. Within one aspect of the invention retroviral vector particle producing cells are provided, wherein the cell (a) has greater than 5, 6, 7, 8, 9, 10, or, 15 stably integrated copies of a retroviral vector construct; (b) produces greater than 5, 10, 20, 50, 75, 100, 150, or, 200 infectious recombinant retroviral vector particles per cell per day; and (c) produces replication incompetent retroviral vector articles. Preferably, such cells will stably produce infectious recombinant retroviral vector particles over at least 30, 50, 75, or, 100 cell doublings, or alternatively, for greater than 2, 3, 4, or, 5 months in cell culture. Within the context of the present invention, xe2x80x9cstable integration of retroviral vector constructsxe2x80x9d refers to integration of the retroviral vector construct into the chromosomal DNA of the host cell. Integration of the retroviral vector construct into chromosomal DNA, and determination of copy number may be readily determined by Southern analysis. Production of replication incompetent retroviral vector particles may be readily determined using the Mus dunni co-cultivation marker rescue assay provided in example 6. Preferably, vector producing cells of the present invention produce no replication competent retrovirus as determined by the above-noted Mus dunni marker rescue assay.
Within various embodiments of the invention, the cell has a stably integrated gag/pol expression cassette, or alternatively, a stably integrated gag expression cassette and a stably integrated pol expression cassette. Further the cell can have a stably integrated env expression cassette.
Also provided by the present invention are methods for producing high titer recombinant retroviral vector particle producing cells, comprising the step of transducing greater than 20, 30, 40, 50, 60, 70, 80, 90, or, 100 recombinant retroviral vector particles per cell into a population of packaging cells. Within another related aspect methods for producing recombinant retroviral vector particle producing cells, comprising transfecting recombinant retroviral vector constructs into a population of packaging cells, wherein at least 5 retroviral vector constructs per cell are stably integrated into said cells. Within certain embodiments, the packaging cell line has a stably integrated gag/pol expression cassette, or alternatively, a stably integrated gag expression cassette and a stably integrated pol expression cassette.
Within yet another aspect of the present invention, methods are provided for producing recombinant retroviral vector particle producing cells, comprising the general steps of (a) generating VSV-G pseudotyped retroviral vector particles; (b) concentrating said particles; and (c) introducing said vector particles into a packaging cell line, such that recombinant retroviral vector particle producing cells are produced.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.